Method of measuring bone resorption activity with a highly enriched population of prefusion osteoclast cells

ABSTRACT

Methods of obtaining enriched populations of osteoclast precursor cells which can be released from tissue culture dishes and used for biochemical studies are described. Osteoblastic cells and bone marrow cells are co-cultured. Next a α v  β 3  receptor ligand, such as echistatin is used for cell detachment. The result is a 75-95% pure enriched population of tartrate resistant acid phosphatase (TRAP + ) cells, in high yields (2-3×10 6  cells per experiment) can be obtained. These cells are mosty mononucleated and based on their characteristics are considered to be pre-fusion osteoclasts (pOC cells). The precursor osteoclasts can be reseeded onto osteoblasts to obtain an enriched population of mature, multinucleated osteoclast cells.

This application is a 35 U.S.C. §371 filing of PCT Application No.PCT/US96/04634, filed Apr. 4, 1996, which is a continuation ofApplication Ser. No. 08/419,210, filed Apr. 10, 1995, now U.S. Pat. No.5,719,058.

DESCRIPTION OF THE INVENTION

This invention relates to a method of producing a population of cellswhich is highly enriched in its content of osteoclast precursor cells,and to the cell populations so produced.

BACKGROUND OF THE INVENTION

Osteoclasts are terminally differentiated cells which play a key role inbone resorption. Due to the low number of mammalian osteoclasts and thedifficulty to isolate them from bone tissue, and from other cells, theircharacterization has been limited primarily to immunohistochemistry oranatomical and physiological measurements on single cells.

Several attempts have been made, with limited success, to identify,isolate and establish in culture, cells capable of differentiating intoosteoclasts from either bone marrow (Billecocq et al., 1990 Pro. Nat.Acad. Sci. USA 87:6470-6474; Prallet et al., 1992, J. Bone Min. Res.7:405-414; and Chambers et al., 1993, Proc. Nat. Acad. Sci. USA90:5578-5582) or leukemic and promyelocytic cell lines (Yoneda et al.,1991, Endocrinology 129:683-689; Gattei et al., 1992 J. Cell Biol.116:437-447). Recently, cells obtained from human giant cell tumors ofbone were reported to form osteoclasts in culture (Grano et al., 1994 J.Bone Min. Res. 9:1013-1020; Grano, et al., 1994 Exp. Cell Res.212:209-218).

To obtain enriched osteoclast preparations, Akatsu et al., 1992, J. BoneMiner. Res. 7:1297-1306; cultured the osteoblastic and bone marrow cellson collagen gel-coated dishes, and released the cells from the collagenmatrix using collagenase. However, the yield of osteoclastic cells wasquite low. Recently, Shioi et al., 1994, Calcif. Tissue Int. 55:387-394;using a similar co-culture system reported the enrichment of generatedosteoclasts by treating the cultured cells with bacterial collagenase toremove the stromal supporting cells. By this procedure they obtained 60%pure tartrate resistant acid phosphate positive (TRAP⁺) cells, howeverthe osteoclasts were not available except as attached cells to eitherdishes or bone slices.

Oursler et al., 1991, J. Bone Min. Res. 6:375-385 cultured avianosteoclasts and further purified the osteoclasts by density gradientcentrifugation. This method yielded an enriched population of TRAP⁺cells capable of bone resorption.

To date, however, the only mammalian osteoclasts obtained in high yields(300,000 per rabbit) and purity (98%) are rabbit osteoclasts (Tezuka etal., 1992, Biochem. Biophys. Res. Comm. 186: 911-917). These cells,however were attached to the plastic culture dishes and cannot beremoved.

DESCRIPTION OF THE INVENTION

This invention relates to a method of obtaining a highly enrichedpopulation of osteoclast precursor cells comprising co-cultivation ofosteoblastic cells and bone marrow cells and treatment of the culturewith an integrin α_(v) β³ receptor ligand to release a population whichis highly enriched with precursor osteoclasts. This invention alsocomprises the process of obtaining a highly enriched population ofmature osteoclast cells comprising reseeding the population of highlyenriched precursor osteoclast cells so produced on osteoblast cells fora time sufficient for the osteoclast precursor cells to fuse into maturemultinucleated osteoclasts.

This invention also relates to the enriched populations of mammalianprecursor osteoclasts. Yet another aspect of this invention is apopulation which is highly enriched with mature osteoclasts. Furtheraspects of this invention is a suspension culture comprising mammalianosteoclast precursor cells, and to suspension cultures comprising matureosteoclast cells.

A further aspect of this invention is an assay for bone resorptionactivity comprising exposing a population of manunalian cells which ishighly enriched in osteoclasts, but also comprising osteoblasts, to abone, in the presence of Vitamin D3 or a biologically active derivativeof Vitamin D3 and measuring the bone-resorption which occurs.

In accordance with this invention, it was observed that integrin α_(v)β₃ receptor ligands appear to play a role in cell attachment and in thefusion of the precursor osteoclasts into multinucleated osteoclasticcells in the bone marrow co-culture system. One aspect of this inventionthus uses α_(v) β₃ receptor ligands to isolate a highly enrichedpopulation of pre-fusion cells. The pre-fusion cells (also referred toas precursor osteoclasts) can be co-cultured with osteoblasts so thatthey differentiate into mature osteoclasts. This invention also relatesto the enriched populations of precursor osteoclasts and matureosteoclasts produced by the processes described above.

As used throughout the specification and claims, the followingdefinitions will apply:

"α_(v) β₃ receptor ligand" is any molecule which binds to the α_(v) β₃receptor. An α_(v) β₃ receptor ligand can be identified by determiningwhether it replaces echistatin which has bound to the α_(v) β₃ receptorin a competitive binding assay. Those compounds which replace echistatinare considered α_(v) β₃ receptor ligands. Examples of α_(v) β₃ receptorligands include RGD-containing peptides and non-peptides which bind tothe same receptor as RGD-peptides.

"highly enriched" means that a population of cells contains at leastabout 75% of a single type of cell, more preferably at least about 90%,and more preferably at least about 95% of a particular cell type.

"biologically active derivative of Vitamin D3" refers to any metaboliteof Vitamin D3 which is normally produced by a mammal and contributes tobone metabolism. Examples include 25-hydroxy Vitamin D3, and1,25-dihydroxy Vitamin D3.

"precursor osteoclasts" are pre-fusion osteoclast cells. Precursorosteoclast cells are characterized by their ability to generatebone-resorbing, giant, multi-nucleated osteoclasts within about 24 hoursof being plated on osteoblasts. Like mature multi-nucleated osteoclasts,they may also be defined by the following characteristics: they areTRAP⁺ ; have calcitonin (CT) receptors; express α_(v) β₃ integrin; andexpress osteopontin (OP) and carbonic anhydrase II (CAII). The precursorosteoclasts made according to the processes of this invention also formpits when plated on bone slices along with osteoblasts in the presenceof Vitamin D3 or a biologically active derivative of Vitamin D3.

"suspension culture" means any culture containing precursor osteoclastsor mature osteoclasts which are not attached to the culture vessel.

Any α_(v) β₃ receptor ligand may be used in accordance with thisinvention. Known α_(v) β₃ receptor ligands include vitronectin,echistatin, kirstin, other snake venoms, other RGD-containing peptides,and non-peptides which bind to the same receptor as RGD-containingpeptides. One preferred α_(v) β₃ receptor ligand is echistatin.Echistatin is an RGD-containing snake venom which inhibits osteoclastattachment and formation (Sato, et al., 1990 J. Cell Biol.111:1713-1723; Tanaka, et al., 1991, J. Bone Min. Res. 6:S148 both ofwhich are hereby incorporated by reference.).

One aspect of this invention is a convenient procedure to obtain asuspension culture of abundant, highly enriched precursor osteoclastcells which (except for their mononucleated characteristic) have all thecharacteristics of mature multinucleated osteoclasts. The cultures ofthis invention comprise a population of cells, at least about 75% ofwhich are precursor osteoclasts. In a preferred embodiment, thepopulation comprises at least about 90% precursor osteoclasts, and inparticularly preferred embodiments, the population contains at leastabout 95% precursor osteoclasts. One important distinguishing feature ofthis invention is that the precursor osteoclasts of the population arein a suspension, i.e. they are not attached to a plastic or glasssubstrate, and are thus able to be further manipulated by theresearcher. This invention thus makes it possible to study thebiochemistry of osteoclasts, the regulation of genes expressed by thesecells and other characteristics.

The culture processes of this invention can be used with any species ofanimal cells which can form osteoclasts, and are able to be cultured invitro. Preferred animals include mammals, particularly those of bovines,rodents (such as mouse and rat), and primates (such as human andmonkey). Particularly preferred are human, and mammalian species such asrat and mouse or other animals whose osteoclasts share biochemicalcharacteristics with human osteoclasts.

This invention includes the first detailed characterization of theproperties of mononuclear cells which can generate multinucletedosteoclasts within 24 hours in culture. While not wishing to be bound bytheory. since the cells of this invention have all the properties testedso far which are attributed to highly differentiated osteoclasts, it isbelieved that precursor osteoclast cells represent a stage ofdifferentiation in the osteoclast lineage cells just prior to fusion,which is the last step in osteoclast differentiation.

In accordance with this invention, bone marrow and osteoblast cells areco-cultured according to known techniques, and using known and availablecell lines. For example, one mouse osteoblast cell line which may beused is MB1.8 (a mouse calvaria-derived osteoblastic cell line), but anyother cell line which supports osteoclastogenesis may also be employed,including ST-2 (a publicly available line), other calvaria-derivedosteoblastic stromal cell lines, or primary osteoblast cells.

Co-culture of bone marrow and osteoblast cells should continue forapproximately 6 days (depending on the particular cell lines used) oruntil the cell lines have yielded the maximum amount of pre-fusionosteoclast cells. However, if cells are cultured too long,multinucleated osteoclasts will form which, due to their attachment tothe cell culture plate, cannot be used in this invention. Prior to theformation of giant multi-nucleated osteoclasts, the majority of cells ofthe osteoblast cell line should be detached from the co-cultured bonemarrow cells using an enzyme system which disrupts the co-culture, suchas dispase/collagenase. The amounts of enzymes may vary with the specifcenzyme used and the specific activity, but should be sufficient to breakup the attachment of osteoblasts to precursor osteoclasts. Generally,approximately 1 mg/ml of each enzyme is sufficient.

Next, the remaining cells are treated with the α_(v) β₃ receptor ligandfor a time and an amount sufficent to inhibit the formation ofmultinucleated osteoclastic cells in the co-culture system as well as toinhibit the attachment and function of multi-nucleated osteoclaststhemselves. In a preferred embodiment of this step, the remaining cellswere treated with 10 to 50 nM echistatin for at least about 10 minutes.In a particularly preferred embodiment, approximately 30 nM ofechistatin is used for approximately 20 minutes.

Cells isolated from bone marrow co-cultures by treatment with the α_(v)β₃ receptor ligand attach to coverslips or tissue culture wells withinone hour. This isolated population of cells is well-defined: based ontheir TRAP⁺ staining and their ability to form mature osteoclasts whenseeded back on osteoblasts, these cells were identified as precursorosteoclasts (pOC cells). Seventy-five to ninety percent of the cells aremononuclear; the rest are multinucleated cells with two to four nuclei.Almost all the cells (88-95%) are TRAP⁺. A few TRAP⁺ cells,(approximately 2-3%) may still be attached to alkalinephosphatase-positive mononuclear cells, presumably osteoblastic cells. Asmall number of cells which (approximately 3-4%) do not stain for eitherTRAP or alkaline phosphatase may also be present.

Another feature of this invention is that a high yield of cells ispossible. For example, yields of about 150,000 cells per 150 cm² tissueculture dish, or approximately 1000 cells/cm² are common.

To further characterize the precursor osteoclast cells so produced,various assays were performed on the highly enriched population. Sincethe presence of abundant calcitonin receptors is regarded as adistinctive marker of the osteoclast phenotype, the pOC cells wereexamined for this property according to known techniques. The osteoclastprecursors bound a large number of salmon calcitonin (¹²⁵ I-sCT)molecules, as visualized by autoradiography, and binding was displacedby a 100 fold excess of unlabeled sCT. However, some TRAP⁺ cells did notbind calcitonin.

Exposure to salmon calcitonin (sCT) for 10 minutes increased cAMP in pOCcells in a dose-dependent manner, up to 250 fold at 10 nM sCT, whilePGE₂ had a small stimulatory effect and PTH was without effect.

The isolated pOC cells, when plated onto 1,25-dihydroxy D₃ -treatedosteoblast cells fuse rapidly (within 24 hours) to form multinucleatedTRAP+ osteoclast-like cells. The pOC cells which are kept frozen inliquid nitrogen and thawed, retain the capability to form multinucleatedTRAP+cells.

The pOC cells possess other features of multinucleated osteoclasts. Onslices of steer cortical bone, and in the presence of osteoblasts andVitamin D3 or a biologically active derivative of Vitamin D3, the pOCcells formed pits after 24 hours of incubation and this activity wasinhibited by calcitonin. The resorption pits were heterogenous in size,varying from single to composite pits. The resorption pits coincidedwith intense F-actin staining. The actin rings could be visualized asearly as 2 hours after the pOC cells are plated on bone slices.

In accordance with this invention, the pOC cells resorb bone only in thepresence of osteoblastic cells and 1,25-dihydroxy vitamin D₃. It wasalso found that the number of pits as well as the number of F-actinrings not only correlate with each other, but both increaseproportionately when pOC cells are co-cultured with an increasing numberof osteoblast cells. There were no F-actin rings when pOC cells werecultured in the absence of osteoblastic cells or a Vitamin D₃derivative.

The pOC cells express mRNAs for proteins found in multinucleatedosteoclasts. For example, high mRNA levels for α_(v) (7.0 kb) and β₃(6.5) integrin subunits, calcitonin receptor (4.2 kb), carbonicanhydrase II (1.8 kb) and osteopontin (1.8 kb) can be observed. Inaddition, they express mRNA for protein tyrosine phosphatase ε (a majortranscript of about 5 kb and a minor transcript of about 3 kb), OC-2(1.8 kb), a possible cysteine proteinase, and matrix metallo-proteinase9 or 92 kD type IV collagenase (3.0 kb). All of these mRNAs are known tobe highly or preferentially expressed in osteoclasts. These findingsillustrate the use of pOC cells produced according to this invention foridentifying and studying osteoclast-associated proteins.

Isolated osteoclasts as well as multinucleated osteoclasts derived fromthe in vitro co-cultures express high levels of pp60^(c-src) pOC celllysates immunoprecipitated with the monoclonal antibody mAB 327, whichrecognizes c-src 60 kDa protein, and immunoblotted with phosphotyrosineantibodies, show an abundance of phosphorylated pp60^(c-src).

The highly enriched population of pOC cells can then be reseeded ontoosteoblast cells in order for the pOC cells to differentiate intomultinucleated osteoclasts. The osteoblastic cells may be the same asused in the co-culture process, or may be a different osteoblast line.An example of a suitable osteoblast line is the murine MB1.8 line whichhas been treated with 1,25-dihydroxy-vitamin D₃. Other appropriate linesinclude: bone marrow stromal lines, primary osteoblast cells lines fromcalvaria or any other cell line which supports osteoclast formation.Upon plating, the pOC cells fuse within a short period of time,generally within 24 hours, to form multinucleated, TRAP⁺ osteoclasts.

Another aspect of this invention is an assay for the bone resorptioninhibitory or stimulatory effect of a test substance comprising placingan osteoclast-enriched population of cells which also comprisesosteoblasts on a bone slice, in the presence of Vitamin D3 or abiologically active derivative thereof, and a test substance (which maycontain a putative inhibitory substance or a putative stimulatorysubstance) and measuring the amount of bone resorption, by, e.g.,determining the number of pits formed. The resulting activity can becompared to the activity of a control where no test substance waspresent. Thus a further aspect of this invention is a method ofidentifying a drug which inhibits bone resorption comprising placing anosteoclast-enriched population of cells which also comprises osteoblastson a bone slice in the presence of Vitamin D₃ or a biologically activederivative thereof, and a putative drug having bone resorptioninhibitory activity, and determining whether resorption occurs. Thisinvention also includes bone resorption-inhibiting drugs identified bythis method.

The following non-limiting Examples are presented to better illustratethe invention.

EXAMPLE 1

Co-Culture And Isolation of Pre-Fusion Cells

Mouse bone marrow-osteoblast co-culture system was performed essentiallyas described by Takahashi et al., 1988 Endocrinology 123:2600-2602,which is hereby incorporated by reference. A cell line (MB1.8), (Tanakaet al., 1992 J. Bone Min. Res. 7:S307) established from neonatal mousecalvaria was found to support bone marrow cell differentiation intoosteoclasts. MB1.8 cells were plated at 10,000/cm² in Alpha Modificationof Minimal Eagle's Medium (α-MEM) containing 10% fetal bovine serum and10 nM 1,25(OH)₂ D₃. (Fetal Bovine Serum was from JRH Biosciences,Lenexa, K. S. Other tissue culture reagents, were from Gibco/BRL LifeTechnologies, Inc., Grand Island, N.Y.)

Balb/C male mice (six weeks old) were sacrificed under CO₂, and tibiaeand femora were aseptically removed. The bone ends were cut off withscissors and the marrow cavity was flushed with 1 ml α-MEM by using a27G needle. The bone marrow cells were then filtered through 70 mm nylonmesh. Cells were centrifuged for 7 minutes at 300×g and washed once withα-MEM and finally resuspended and aliquoted at 25,000 cells/cm² onto theMB1.8 cultures. Medium was replaced every two days.

After 6 days, many mononuclear and some multinuclear TRAP+ cells werepresent in these cultures. Cultures were washed twice with PBS andtreated with collagenase/dispase (1 mg/ml each in PBS) at 37° C. for 20minutes. (Collagenase Cat. # 034-10533 was from Wako Chemicals USA,Inc., Dallas, Tex. Dispase Cat. # 165859 was from Boehringer MannheimCorp., Indianapolis, Indi.) Released cells, mostly osteoblasts, wereremoved with a pipet and the plates were washed three times with PBS.The remaining cells were incubated with 30 nM echistatin in α-MEMcontaining 1% BSA for 20 minutes at 37° C. These cells were collected,pelleted and washed once with α-MEM containing 10% FBS and were platedeither on bone, coverslips or in wells, as indicated below.

                  TABLE 1                                                         ______________________________________                                        Abundance of TRAP.sup.+ cells in enriched populations of pOC cells              Experiment number TRAP.sup.+ cells                                                                          TRAP.sup.- Cells                              ______________________________________                                        1               829 (94%)    48 (6%)                                            2 614 (88%)  81 (12%)                                                         3 1006 (91%)  102 (9%)                                                      ______________________________________                                    

Freshly isolated pOCs were plated at 12,500 cells/cm² on serum coatedwells and allowed to attach for 2 hrs. Cells were fixed and stained forTRAP and in some experiments counterstained for alkaline phosphatase asdescribed. Approximately 1,000 cells were counted per well.

EXAMPLE 2

Characterization of pOC Cells

Measurement of cAMP: Osteoclast precursors were isolated as described inExample I and plated at 240,000 cells per well in 24 well dishes thatwere precoated with FBS. Cells were allowed to attach for 90 minutes,washed twice with α-MEM and treated with 1 mM isobutylmethylxanthine(IBMX) for 10 minutes at 37° C. Bovine parathyroid hormone (PTH), salmoncalcitonin (sCT), or PGE₂ were added at the indicated concentrations andthe cultures were incubated for 10 minutes at 37° C. (Bovine PTH 1-34was from Bachem, Inc., Torrance, Calif. Salmon calcitonin was fromPeninsula Laboratories. Belmont, Calif. The IBMX and TRAP kit were fromSigma, St. Louis, Mo.).

The medium was aspirated and the cells were extracted three times withabsolute ethanol. The extracts were evaporated to dryness and analyzedfor cAMP by radioimmunoassay according to the manufacturer'sinstructions (Amersham Corp.).

                  TABLE 2                                                         ______________________________________                                        Effects of CT, PTH and PGE.sub.2 on cAMP accumulation in pOC cells                        Additions  cAMP (pmol/10.sup.5 cells)                             ______________________________________                                        Experiment 1                                                                              none       0.29 ± 0.026                                           sCT(10.sup.-11 M) 6.81 ± 1.14*                                             sCT(10.sup.-8 M) 70.8 ± 5.1*                                              Experiment 2 none 0.15 ± 0.025                                              sCT (10.sup.-8 M) 36.9 ± 2.5*                                              PTH (10.sup.-7 M) 0.19 ± 0.027                                             PGE.sub.2 (10.sup.-6 M)  0.27 ± 0.008*                                  ______________________________________                                    

Freshly isolated pOCs were plated on serum coated 24-well culture dishesat 120,000 cells/cm² (Exp. 1) or 65.000 cells/cm² (Exp. 2) and allowedto attach for 2 hours. Cultures were washed and pretreated for 10 minsat 37° C. with 1 mM IBMX. Cells were then incubated for 10 mins withsCT, PTH or PGE₂ at 37° C. The medium was aspirated and cAMP wasextracted 3× with ethanol. The extracts were analyzed for cAMP byradioimmunoassay as described. Results given in Table 2, above are themean±s.d. for triplicate wells. The asterisk signifies that the amountwas significantly greater than control group, p<0.001.

Calcitonin Autoradiography

Isolated osteoclast precursors from Example 1 were plated on coverslipsthat were precoated iwith FBS for two hours at 37° C. The cells wereincubated for one hour at room temperature in α-MEM containing 0.1% BSAand 0.3 nM ¹²⁵ I-sCT with or without 100 fold excess unlabeled sCT. (¹²⁵I-salmon calcitonin (2000 Ci/mmol) was purchased from Amersham Corp.,Arlington Heights, Ill.)

Cultures were washed five times with ice cold α-MEM, fixed in 10%formalin for 10 minutes at room temperature and permeabilized withethanol/acetone (1:1) for 2 minutes and stained for TRAP. Coverslipswere mounted on slides, dipped in ILFORD K.2 emulsion (Polysciences,Warrington, Pa.) diluted 2 parts emulsion:l part 6% glycerol, dried andstored at 4° C. for 2 weeks. Slides were developed in Kodak D-19developer diluted 1:1 with water for 5 minutes followed by fixer for 5minutes, washed with deionized water for 15 minutes and air dried.

RNA Isolation and Northern Blot Analysis

Total cellular RNA was isolated by guanidinium isothio-cyanate andphenol extraction as described in Chomczynski, P et al., 1987 Anal.Biochem. 162:156-159, which is hereby incorporated by reference. TotalRNA (25 μg) was electrophoresed through 1% agarose-formaldehyde gels andelectroblotted onto nylon filters (Hybond-N, Amersham Corp., ArlingtonHeights, Ill.). Blots were prehybridized in buffer containing 50%formamide, 5×SSC (1×SSC=0.15 M NaCl, 0.015 M sodium citrate),5×Denhardt's solution and 100 μg/ml sonicated salmon sperm DNA andhybridized at 42° C. in fresh buffer containing the indicated cDNAswhich were labeled with a random primer DNA labeling kit (PharmaciaBiotech Inc., Piscataway, N.J.) using [α-³² P-dCTP] (Amersham Corp.).Blots were washed with 0.1×SSC/0.1% SDS at 65° C. for 30 minutes andexposed to Kodak XAR film. The cDNA clones were generated for MMP-9,OC-2, and osteopontin by using cDNA libraries of rabbit osteoclasts(Tezuka et al., 1992, Biochem. Biophys. Res. Comm. 186: 911-917; 1994 J.Biol. Chem 269: 15006-15009; and 1994 J. Biol. Chem. 269: 1106-1109 eachof which is hereby incorporated by reference). PTP ε was cloned frommouse osteoclastic cells (Schmidt et al., 1993, J. Bone Min. Res.8:S144, which is hereby incorporated by reference). The following cDNAprobes were cloned by PCR based on the reported sequences: human a_(v)(Suzuki et al., 1987 J. Biol. Chem. 262:14080-14085, which is herebyincorporated by reference; mouse calcitonin receptor (Lin et al., 1991Science 254:1022-1024, which is hereby incorporated by reference); andhuman β₃ (Frachet et al., 1990 Molec. Bio. Rep. 14:27-33, which ishereby incorporated by reference). mAB 327 which recognizes pp60^(c-src)was a gift of Dr. J. Brugge (Lipsich, et al., 1983 J. Virology48:352-360, which is hereby incorporated by reference).

Measurement of Bone Resorption

Bone slices (20 mm²) were prepared from bovine cortical bone by a lowspeed diamond saw (Buehler, Lake Bluff, Ill.). Slices were cleaned byultrasonication (Branson, Shelton, Conn.) in distilled water (15 mins)three times, rinsed in distilled water and placed in 96-well cultureplates (Costar Co., Cambridge, Moss.). The bone slices were sterilizedunder UV light. Bone slices were rehydrated with medium 199 containing10% FBS and penicillin/streptomycin solution. A suspension (20,000cells) of the TRAP⁺ bone marrow cells were added to each well with orwithout MB1.8 cells in the presence or absence 10 nM1,25-dihydroxy-Vitamin D₃, or in the presence or absence of MB1.8 cellspre-treated for 48 hours or not with 1,25-dihydroxy Vitamin D₃(1,25(OH₂)D₃). After 24 hrs, the bone slices are fixed and stained with1% toluidine blue as described previously (Demster, et al., 1987 J. BoneMin. Res. 2:443-448; 1987.). Results are given in TABLE 3, below.

                  TABLE 3                                                         ______________________________________                                        Effects of osteoblastic cells and 1,25-dihydroxy - Vitamin D.sub.3 on         bone                                                                            resorption by pOC cells                                                                      Number of pits/bone slice                                    Culture Conditions                                                                             Experiment 1                                                                             Experiment 2                                      ______________________________________                                        pOC cells        0          0                                                   + 1,25 (OH).sub.2 D.sub.3 0 1                                                 + MB1.8 cells ND 1                                                            + MB1.8 cells + 1,25(OH).sub.2 D.sub.3 ND 56 ± 20                          Pretreated with 1,25(OH).sub.2 D.sub.3 :                                      + MB1.8 cells 178 ± 59 57 ± 40                                          + MB1.8 cells + 1,25(OH).sub.2 D.sub.3 387 ± 60 196 ± 68              ______________________________________                                         ND = not determined                                                      

Immunofluorescence Microscopy

Bone slices containing the pOC cells in the presence or absence of MB1.8cells or 1,25-dihydroxy Vitamin D₃ (under the same conditions asdescribed above for measurement of bone resorption) were fixed in 3%paraformaldehyde, 2% sucrose in PBS for 5 min at room temperature. Thecells were permeabilized in 0.5% Triton X-100 for 5 min on ice. F-actinwas stained with rhodamine-conjugated phalloidin (Molecular Probes,Inc.), 5U/ml for 15 min at room temperature.

Immunoprecipitation and Immunoblots

The pOC cells from Example I were allowed to attach to dishes for 3hours and lysed in RIPA buffer (150 mM NaCl, 10 mM Tris, 1% TritonX-100, 1% deoxycholate, 1 mM EDTA, 1 mM sodium orthovanadate, 10 mg/mlleupeptin, 1 TIU/ml aprotinin, and 1 mM PMSF, pH 7.3) 100 μl/100,000cells. The lysate was preincubated with sepharose 4B-200 for 1 hr at 4°C., then divided into 2 aliquots. One was incubated with 2 μlpp60^(c-src) antibody (mAb327) overnight at 4° C., the other was leftuntreated. The lysates were reacted with goat-anti-mouse sepharose for 1hr at 4° C., the beads were washed 5 times with RIPA buffer, thentreated with Laemmli sample buffer for 3 minutes at 95° C. and run on a10% SDS polyacrylamide gel (Laemmli, 1970 Nature 227:680-685, which ishereby incorporated by reference). The proteins were electro-transferredto Immobilon P overnight, the membrane was blocked in 100 mM NaCl, 10 mMTris, 0.1% Tween, 1% BSA, incubated with anti-phophotyrosine antibodyhorseradish peroxidase conjugate RC20H (Transduction Laboratories,Lexington, Ky.) diluted 1:10,000, washed extensively, reacted with ECLreagents (Amersham) and exposed to XAR5 film (Kodak). Each lanecontained protein from 100,000 cells.

What is claimed is:
 1. An assay for the bone resorption inhibitory orstimulatory effect of a test substance comprising: placing a highlyenriched population of tartrate resistant acid phosphate positive(TRAP+) prefusion osteoclast cells in suspension culture, saidpopulation also comprising osteoblasts which support osteoclastogenesis,on a bone slice in the presence of Vitamin D3 or a biologically activederivative thereof and the test substance, and measuring the amount ofbone resorption.
 2. An assay according to claim 1 wherein the amount ofbone resorption measured is compared to a result obtained in the absenceof a test substance.
 3. A method for identifying a drug which inhibitsbone resorption comprising: placing a highly enriched population oftartrate resistant acid phosphate positive (TRAP+) prefusion osteoclastcells in suspension culture, said population also comprising osteoblastswhich support osteoclastogenesis on a bone slice in the presence ofVitamin D3 or a biologically active derivative thereof, and a putativedrug having bone resorption-inhibitory activity, and determining whetherresorption occurs.
 4. An assay in accordance with claim 1 wherein theosteoclast cells are mammalian.
 5. An assay in accordance with claim 1wherein the osteoclast cells are selected from the group consisting ofhuman, mouse and rat cells.
 6. An assay in accordance with claim 3wherein the osteoclast cells are mammalian.
 7. An assay in accordancewith claim 3 wherein the osteoclast cells are selected from the groupconsisting of human, mouse and rat cells.